Project Summary: This revised U01 research project is in response to PAR-16-214 with the Funding Opportunity Title, ?Program for Extramural/Intramural Alcohol Research Collaborations?. Sulie L. Chang, Ph.D. and Ming D. Li, Ph.D. will be the two extramural investigators at Seton Hall University, and David Goldman, M.D. will be the intramural investigator at NIAAA/NIH. For their alcohol research collaborations, they will share their unique, yet complementary, expertise and experiences as well as their research facilities and other resources. The project goal is to delineate the involvement of DNA methylation in binge ethanol (EtOH)-induced spleen atrophy during adolescence. Adolescence is an important period for maturation of various physiological functions, including immune responses. Binge alcohol drinking, defined as consumption of excessive amounts of alcohol in a short time resulting in a blood EtOH concentration (BEC) of at least 0.08 g/dl, is popular among adolescents and can lead to addictive behaviors and eventually alcoholism in adulthood. Atrophy of the spleen, a key immune organ, is highly associated with immune dysfunction. We have shown that treatment with 4.8 g/kg/d EtOH for 3 d differentially decreases the size of the spleen in 5 wk old adolescent male F344 rats, but not in adult rats. There was also a decrease in the area of the splenic white pulp and a distortion of white pulp structure in Sprague Dawley rats binge treated with EtOH. Expression of CD3, a T cell marker, was decreased. The CD3+ T cell population was also differentially decreased in F344 rats given binge EtOH. These data showed that loss of T cells might partially, account for distortions of white pulp and the overall relative spleen weight. The expression of caspase-3, a key enzyme for cell death, negatively correlated with the relative spleen weight, indicating that binge EtOH induces loss of T cells leading to distortions of the white pulp and spleen atrophy, in part, due to apoptosis. DNA methylation is an epigenetic event that operates through post-transcriptional modification of DNA by DNA methyltransferases (DNMTs) to regulate gene expressions. One of our preliminary studies showed that treatment with 5-Aza-2?-deoxycytidine (5-AZ), an inhibitor of DNA methylation, reversed binge EtOH-induced spleen atrophy. In parallel, we also found that significant decreases of DNMT1 expression in the spleens of rats given binge EtOH. The qRT-PCR array data showed that 6 genes were significantly downregulated among 84 immunity and EtOH metabolism related genes after binge drinking. Taken together, we hypothesize that DNA methylation is involved in binge EtOH-induced regulation of various target genes leading to apoptosis of T cells, distortion of white pulp, and spleen atrophy during adolescence. To test this hypothesis, in this revised application, we propose the following three aims: Aim 1 is to investigate the effects of binge EtOH consumption on spleen atrophy, and to isolate and characterize CD3+ cells for the studies in Aims 2 and 3. Aim 2 is to investigate the role of methylation in binge EtOH-induced spleen atrophy using genome bisulfite sequencing (WGBS), followed by genome-wide methylation analysis (GWMA). Aim 3 is to determine the involvement of methylation in binge EtOH-induced spleen atrophy at the gene expression and mechanistic levels by studying the expression of methylated genes, including the known EtOH regulated genes (KERGs) and WGBS identified methylated genes (WGIMGs). In addition, the methylation inhibitor, 5-AZ, will be used to confirm if methylation of these identified genes (e.g.,methylated KERGs and WGIMGs) is involved in binge-EtOH induced expression of these genes. The proposed studies have combined data-driven discovery and hypothesis-driven investigation together. We expect to identify whole-genome methylated loci in the spleens of adolescent rats subjected to a binge EtOH regimen and the methylation loci of the KERGs and WGIMGs and determine the expression and activities of the proteins encoded by these genes. With these information, we will be able to develop epigenetic-based interventions to curtail binge alcohol-induced spleen injury during adolescence.